Carbonic anhydrase having increased stability under high temperature conditions

ABSTRACT

The present invention relates to polynucleotide and polypeptide sequences of novel carbonic anhydrase variants having increased stability under high temperature conditions compared to native carbonic anhydrase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of prior application Ser. No. 11/409,487 filed Apr. 20, 2006, now U.S. Pat. No. 7,521,217, which claimed priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/673,345 filed on Apr. 21, 2005.

FIELD OF THE INVENTION

The present invention relates to polynucleotide and polypeptide sequences of novel carbonic anhydrase variants having increased stability under high temperature conditions compared to native carbonic anhydrase.

BRIEF DESCRIPTION OF THE PRIOR ART

Carbonic anhydrase (EC 4.2.1.1.) is a globular zinc metalloenzyme of molecular weight 30,000 daltons. The enzyme was discovered in 1933 and has been the subject of intense scientific investigation. Multiple isoforms have been discovered in plant and animal tissues where it is believed to facilitate the transport of carbon dioxide. Red blood cells contain isoenzymes I and II, which are the most active. Carbonic anhydrase II has the highest molecular turnover number of any known enzyme. One molecule of carbonic anhydrase can hydrate 36,000,000 molecules of carbon dioxide in a period of 60 seconds. Physiologically, carbonic anhydrase facilitates the removal of carbon dioxide from the mammalian body. The general enzyme reaction is shown below. CO₂+H₂O

H⁺+HCO₃ ⁻

Human carbonic anhydrase II (CAII) variants have also been the subject of scientific investigation. Indeed, the functional importance of a conserved hydrophobic face in human carbonic anhydrase II (CAII), including amino acid residues 190-210, was investigated by random mutagenesis.¹

Other CAII variants have been obtained by substituting amino acids of varying size at position 65, for instance by changing the amino acid Ala for the amino acid Thr. This modification was done in order to investigate the importance of maintaining the active site water network for efficient proton transfer.^(2,3)

A library of CAII variants differing in hydrophobic amino acid residues Phe93, Phe95, and Trp97 was also prepared using cassette mutagenesis, then displayed on filamentous phage, and screened for proteins retaining high zinc affinity.⁴

It exists a need in the art for the development of innovative carbonic anhydrase variants harboring advantageous characteristics over native carbonic anhydrases, such as exhibiting increased stability under high temperature conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the residual enzymatic activity of modified carbonic anhydrases II according to preferred embodiments of the invention following a 2 hour treatment at 55° C.

FIG. 2 shows the residual enzymatic activity of modified carbonic anhydrases II according to preferred embodiments of the invention following a 2 hour treatment at 60° C.

FIG. 3 shows the residual enzymatic activity of modified carbonic anhydrases II according to preferred embodiments of the invention following a 2 hour treatment at 62.5° C.

FIG. 4 shows the residual enzymatic activity of modified carbonic anhydrases II according to preferred embodiments of the invention following a 2 hour treatment at 65° C.

FIG. 5 shows the residual enzymatic activity of modified carbonic anhydrases II according to preferred embodiments of the invention following a 2 hour treatment at 70° C.

FIG. 6 shows the amino acid sequence of the native unmodified carbonic anhydrase 11 (SEQ ID NO: 1).

FIGS. 7 to 16 show the amino acid sequence of modified carbonic anhydrases according to preferred embodiments of the invention (SEQ ID NOS: 2 to 11).

FIGS. 17 to 26 show the nucleotide sequence encoding the carbonic anhydrases of FIGS. 7 to 12 (SEQ ID NOS: 12 to 21).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have surprisingly found that a number of mutations of human carbonic anhydrase II (HCAII), individually or in combination, provide a stabilizing effect on the modified HCAII protein and enable enzymatic activity at higher temperature than normal (i.e higher than 25° C.). In this connection, the present invention specifically relates to the identification of polypeptides and polynucleotide sequences encoding a modified carbonic anhydrase (CA), preferably of human origin, which have increased stability compared to native CA.

1. Definitions

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

As used herein, the expression “high temperature conditions” refers to temperature higher than 25° C. and lower than 70° C. Preferably, it refers to temperature higher than about 37° C., more preferably higher than about 55° C. and even more preferably higher than about 65° C. By “about”, it is meant that the value of said temperature can vary within a certain range depending on the margin of error of the method or apparatus used to evaluate such temperature. For instance, the margin of error may range between ±1° C. to ±5° C.

As used herein, the term “polypeptide(s)” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. “Polypeptide(s)” refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers, and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. “Polypeptide(s)” include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide.

A “functional derivative”, as is generally understood and used herein, refers to a protein/peptide/polypeptide sequence that possesses a functional biological activity that is substantially similar to the biological activity of the whole protein/peptide/polypeptide sequence. In other words, it refers to a polypeptide of a modified CA of the invention that substantially retain(s) the capacity of catalyzing the hydration of carbon dioxide. A functional derivative of a modified CA protein/peptide of the invention may or may not contain post-translational modifications such as covalently linked carbohydrates, if such modification is not necessary for the performance of a specific function. The term “functional derivative” is meant to encompass the “fragments” or “chemical derivatives” of a modified CA protein/peptide of the invention. As used herein, a protein/peptide is said to be a “chemical derivative” of a modified CA protein/peptide of the invention when it contains additional chemical moieties not normally part of the protein/peptide, said moieties being added by using techniques well known in the art.

By “substantially identical” when referring to a polypeptide, it will be understood that the polypeptide of the present invention preferably has an amino acid sequence having at least 80% identity, or even preferably 85% identity, or even more preferably 95% to SEQ ID NOS:1 to 11, or functional derivatives thereof.

One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or homology for an optimal alignment. A program like BLASTp will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated by the present invention.

With respect to protein or polypeptide, the term “isolated polypeptide” or “isolated and purified polypeptide” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated and modified polynucleotide molecule contemplated by the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.

The term “substantially pure” refers to a preparation comprising at least 50-60% by weight of the modified CA polypeptide of the invention. More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, of the modified CA polypeptide of the invention.

Purity is measured by methods appropriate for the modified CA polypeptide of the invention (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

As used herein, the term “polynucleotide(s)” generally refers to any polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. This definition includes, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, cDNA, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. The term “polynucleotide(s)” also embraces short nucleotides or fragments, often referred to as “oligonucleotides”, that due to mutagenesis are not 100% identical but nevertheless code for the same amino acid sequence.

By “substantially identical” when referring to a polynucleotide, it will be understood that the polynucleotide of the invention has a nucleic acid sequence which is at least 65% identical, more particularly 80% identical and even more particularly 95% identical to any one of SEQ ID NO 12 to 21 or functional fragments thereof.

A “functional fragment”, as is generally understood and used herein, refers to a nucleic acid sequence that encodes for a functional biological activity of protein that is substantially similar to the biological activity of protein coding of the whole nucleic acid sequence. In other words, it refers to a nucleic acid or fragment(s) thereof that substantially retains the capacity of encoding a carbonic anhydrase polypeptide of the invention.

The term “fragment”, as used herein, refers to a polynucleotide sequence (e.g., cDNA) which is an isolated portion of the subject nucleic acid constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art.

With reference to polynucleotides of the invention, the term “isolated polynucleotide” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′directions) in the naturally occurring genome of the organism from which it was derived. For example, the “isolated polynucleotide” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote. An “isolated polynucleotide molecule” may also comprise a cDNA molecule.

Amino acid or nucleotide sequence “identity” and “similarity” are determined from an optimal global alignment between the two sequences being compared. An optimal global alignment is achieved using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453). “Identity” means that an amino acid or nucleotide at a particular position in a first polypeptide or polynucleotide is identical to a corresponding amino acid or nucleotide in a second polypeptide or polynucleotide that is in an optimal global alignment with the first polypeptide or polynucleotide. In contrast to identity, “similarity” encompasses amino acids that are conservative substitutions. A “conservative” substitution is any substitution that has a positive score in the blosum62 substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919). By the statement “sequence A is n % similar to sequence B”, it is meant that n % of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides and conservative substitutions. By the statement “sequence A is n % identical to sequence B”, it is meant that n % of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides.

2. Modified Ca Polynucleotides and Polypeptides of the Invention

In a first embodiment, the present invention concerns a modified carbonic anhydrase polypeptide having increased stability under high temperature conditions compared to unmodified carbonic anhydrase, i.e. a modified CA that satisfactory retains enzymatic activity at a temperature higher than suitable for use with native CA (for instance higher than about 25° C.). As used herein, the term “modified CA” refers to forms of CA that differ structurally from unmodified CA. In particular, the modified CA protein of the invention comprises an amino acid sequence substantially identical to SEQ ID NO 1 and wherein the modified CA comprises at least one amino acid substitution at a position, or at an equivalent position, corresponding to position 65, 93, 100, 136, 153, 197, 223, 239 and 247 of SEQ ID NO 1. In this connection, the term “equivalent position” denotes a position which, on the basis of an alignment of the amino acid sequence of the parent carbonic anhydrase in question with the “reference” carbonic anhydrase amino acid sequence in question (for example the sequence shown in SEQ ID No. 1) so as to achieve juxtapositioning of amino acid residues/regions which are common to both, corresponds most closely to a particular position in the reference sequence in question.

The substituted amino acid is selected such that and as previously mentioned, the modified CA retains catalytic activity (i.e. the interconversion of CO₂ with HCO₃— and H+) and exhibits increased stability compared to unmodified CA. The term “substituted amino acid” is intended to include natural amino acids and non-natural amino acids. Non-natural amino acids include amino acid derivatives, analogues and mimetics. As used herein, a “derivative” of an amino acid refers to a form of the amino acid in which one or more reactive groups on the compound have been derivatized with a substituent group. As used herein an “analogue” of an amino acid refers to a compound that retains chemical structures of the amino acid necessary for functional activity of the amino acid yet also contains certain chemical structures that differ from the amino acid. As used herein, a “mimetic” of an amino acid refers to a compound in that mimics the chemical conformation of the amino acid.

Preferred amino acid substitutions consist of Ala65Thr, Phe93Leu, Leu100His, Gln136Tyr, Gln136His, Lys153Leu, Leu197Met, Leu223Ser, Leu239Pro or Ala247Thr. More particularly, the modified CA of the invention comprises an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS: 2 to 11 or functional derivatives thereof.

It will be understood that while the modified CA of the invention may comprises only one amino acid substitution at a position, or at an equivalent position, corresponding to position 65, 93, 100, 136, 153, 198, 223, 239 and 247 of SEQ ID NO 1, it may be advantageous to provide a modified CA which comprises a combination of any of the amino acid substitution mentioned above. In other words, the present invention also advantageously concerns a modified CA protein that comprises a combination of two (2×), three (3×), four (4×), five (5×), six (6×), seven (7×), eight (8×) or of nine (9×) of the amino acid substitutions mentioned above. Preferred combinations contemplated by the present invention are those shown in Table 1.

In another embodiment, the present invention concerns an isolated polynucleotide encoding a modified CA polypeptide of the invention. Preferably, the isolated polynucleotide of the invention comprises a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 12 to 21 and functional fragments thereof.

2. Vector

In another embodiment, the invention is further directed to a vector (e.g. cloning or expression vector) comprising a polynucleotide sequence of the invention.

As used herein, the term “vector” refers to a polynucleotide construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, “cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, “expression vectors” which are designed for transcription of a nucleotide sequence in a host cell, or a “viral vector” which is designed to result in the production of a recombinant virus or virus-like particle, or “shuttle vectors”, which comprise the attributes of more than one type of vector.

A number of vectors suitable for stable transfection of cells and bacteria are available to the public (e.g. plasmids, adenoviruses, baculoviruses, yeast baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses), as are methods for constructing such cell lines. It will be understood that the present invention encompasses any type of vector comprising any of the polynucleotide molecule of the invention.

3. Cells

In a further embodiment, the invention is also directed to a host, such as a genetically modified cell, comprising any of the polynucleotide sequence according to the invention and more preferably, a host capable of expressing the polypeptide encoded by this polynucleotide. Even more preferably, the present invention is concerned with a host cell that incorporates an expression vector or a recombinant viral vector as defined herein below.

The host cell may be any type of cell (a transiently-transfected mammalian cell line, an isolated primary cell, or insect cell, yeast (Saccharomyces cerevisiae or Pichia pastoris), plant cell, microorganism, or a bacterium (such as E. coli).

4. Uses of Modified Ca

The modified CA proteins of the invention retain the catalytic activity of unmodified CA. Accordingly, the modified CA proteins are useful for catalysing CO₂. Moreover, since a modified CA protein of the invention has increased stability under high temperature compared to unmodified CA, a particular amount of this modified CA protein exhibits greater catalytic activity over time than an equal amount of unmodified CA.

The modified CA proteins of the invention can also be used in processes such as those described in the following Canadian references: 2.291.785; 2.329.113 and 2.393.016.

In addition to the foregoing uses, the modified CA proteins of the invention, because of their enhanced stability, are particularly well-suited for removing CO₂ from a CO₂ containing effluent, such as a gaseous or liquid effluent, and more particularly for transforming CO₂ in high temperature gaseous effluent such as industrial gaseous effluents.

EXAMPLES

The present invention will be more readily understood by referring to the following examples. These examples are illustrative of the wide range of applicability of the present invention and are not intended to limit their scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.

Example 1 Cloning of Wild-Type Human Carbonic Anhydrase II (HCAII)

Human lymphocytes were isolated from 8 ml of blood, which 7 ml of PBS buffer (phosphate buffer 20 mM, pH 7.4, 150 mM NaCl) was added. This blood/PBS solution was then poured onto a 10 ml Ficoll cushion (Ficoll-Paque, Pharmacia) and centrifuged during 45 minutes at 1900 rpm/22° C. (Megafuge 1.0R, Heraeus instruments). The lymphocytes layer was washed in PBS and then centrifuged at 1900 rpm during 10 minutes to obtain a pellet. The pellet was then resuspended in TRI-Reagent (Molecular Research inc.) was then added to the pellet for RNA isolation. mRNAs were used to form a cDNA by the SuperScriptII reverse transcriptase (Gibco/BRL) according to the manufacturer's instruction. The oligonucleotide used for the reverse transciption was SEQ ID NO: 22 5′ TTTTTTTTTTTTNV 3′.

HCAII cDNA was amplified by PCR with the following specific oligonucleotides:

SEQ ID NO: 23 5′ ATGTCCCATCACTGGGGGTAC 3′ SEQ ID NO: 24 5′ TTATTTGAAGGAAGCTTTGATTTGC 3′.

The amplification was carried out in a thermocycler (Applied Biosystems model 9700) according to the following program: denaturation at 94° C.×2 min followed by 30 amplification cycles: 94° C.×30 sec, 48° C.×45 sec, 72° C.×60 sec and a final extension of the products at 72° C.×7 min. The PCR product was TA-cloned in the pCR2.1 vector (Invitrogene) according to the manufacturer's instructions. The ligation products have been introduced into competent E. coli DH5α (Gibco/BRL) and the transformants were selected on LB-ampicillin agar (100 μg/mL of ampicillin; 80 mg/L X-gal and 0.2 mM IPTG).

The HCAII sequence integrity has been confirmed by direct and reverse sequencing. The coding sequence has been cloned in the expression vector pET28a(+) (NOVAGEN) to provide the pET28a+HCAII vector. Competent E. coli BL21 λDE3 pLysS have been transformed with the ligation products and the transformants were selected on LB agar with kanamycin (30 μg/mL). HCAII synthesis by transformed E. coliλDE3 pLysS in LB broth has been initiated at OD₆₀₀ ˜0.6 by adding IPTG (0.4 mM) and ZnSO₄ (0.5 mM). Synthesis was maintained at 37° C. during 4h00 with an agitation of 250 rpm. Recombinant carbonic anhydrase production was confirmed by SDS-PAGE.

Example 2 Preparation of Modified Carbonic Anhydrases by Random Mutagenesis by PCR

Mutagenic amplification of the HCAII coding sequence was preformed with the pET28a+HCAII vector. The following oligonucleotides were used:

SEQ ID NO: 25 5′ CAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAT ACCGTGGTAATG 3′ SEQ ID NO: 26 5′ GGCTTGCCTGGTGCTCGAGTCATTA 3′ These primers hybridize to the extremities of the HCAII coding sequence. The first primer hybridises upstream to the HCAII coding sequence to the start codon (ATG) and contains a Xbal restriction site. The second primer hydridises downstream to the HCAII coding sequence to the stop codon (TAA) and contains a Xhol restriction site. The restriction sites Xbal and Xhol are useful during the cloning steps of the amplicons, as better detailed hereinafter.

The amplification reaction mixture consist of TRIS-HCl pH 8.3 at 10 mM (Sigma), KCl (Sigma) 50 mM, MnCl₂ (Sigma) 0.15 mM, gelatine 0.01% (Biorad), ATP 2 mM, GTP 2 mM, TTP 10 mM, CTP 10 mM (dNTP Invitrogene), primers 0.5 mM, Taq DNA polymerase 0.5 Units (Promega), plasmid 4 ng in 50 μL. The amplification was performed under the following program: first cycle: 95° C.×2 min., 55° C.×30 sec and 72° C.×30 sec and 14 cycles of 95° C.×30 sec., 55° C.×30 sec and 72° C.×30 sec.

The PCR products and the pET28a(+) vector were enzymatically cut by XbaI et XhoI restriction enzymes (Roche), and the resulting fragments were purified with the QIAquick DNA extraction gel kit (Qiagen) and ligated with T4 DNA ligase (Invitrogen). The recombinant molecules were then introduced by transformation in competent E. coli BL21 λDE3 pLysS. Transformed bacteria were grown on 20 cm×20 cm petri dishes containing LB agar with kanamycin (30 μg/mL) and chloramphenicol (34 μg/mL). Bacteria were incubated at 37° C. overnight. The petri dishes harbouring approximatively 2000-3000 UFC were then photographed.

Example 3 Mutant CA Selection Arboring Increased Carbonic Anhydrase Stability

Colony Transfert

The selection method of thermostabilized carbonic anhydrase was developed according to the method developed by Krebs and Fierke. Following transformation, E. coli colonies were transferred onto a 20 cm×20 cm nitrocellulose filter (Hybon-C Extra; Amersham). The nitrocellulose filter was then disposed with the colonies facing up, onto a second agar containing IPTG (2 mM) and ZnSO₄ (0.5 mM) for initiating synthesis of carbonic anhydrase recombinants. The induction was maintained during four hours at 37° C. The membrane was then placed at −80° C. for at least 30 minutes. The original agar was kept at 4° C. and the membrane was used for the enzymatic assays as described hereinafter.

Membrane Preparation for Enzymatic Assays

The membrane is defrosted at room temperature for 10 minutes. The membrane frost-defrost process induces cellular breakings during the defrosting period which support the release of the cellular contents, such as the lysosyme coded by the pLysS plasmid. This lysosyme causes the lysis of the bacteria and thus, the release of the cytoplasmic contents of the bacteria. The nitrocellulose strongly maintains the released proteins by electrostatic interactions, such as the HCAII (Human Carbonic Anhydrase II) locally over-expressed by the transferred colony. The unoccupied sites on the membrane are blocked using a 100 mM TRIS (Sigma)/10 mM NaCl (Sigma) pH 8.0/5 w.v % powder skimmed milk (Nestle) solution, during the first 10 minutes without any agitation and then 50 minutes using a 50 RPM agitation at room temperature. Then the membrane is washed five times using a 100 mM TRIS/10 mM NaCl pH 8.0 solution (1×15 minutes and 4×5 minutes). After the second 5-minute washing, the fragments of the colonies which have remained linked are removed softly using a gloved finger. After the last washing, a 25 mL pipette is rolled upon the membrane surface using an adequate pressure in order to expel a maximum quantity of buffer from the membrane. Finally, the membrane is soaked in a 25 mM TAPS (Sigma)/100 mM Na₂SO₄ (Fisher) pH 8.4 buffer compatible with enzymatic assays.

Example 4 CA Activity Detection

The selection method uses the hydration activity of CO₂ from the HCAII

$\begin{matrix} {{{Residual}\mspace{14mu}{activity}\mspace{14mu}(\%)} = {\left( \frac{{Activity}\mspace{14mu}{after}\mspace{14mu}{treatment}}{{Activity}\mspace{14mu}{before}\mspace{14mu}{treatment}} \right) \times 100.}} & \left( {{Equation}\mspace{14mu} 1} \right. \end{matrix}$ The hydration reaction of CO₂ releases a proton, leading to a local acidification of the membrane, more specifically where an active HCAII is located. The membrane is coloured using the following buffer: 25 mM TAPS/100 mM Na₂SO₄ pH 8.4/10 mM purple m-cresol (Fisher). Then, the membrane is placed in a container designed specifically for this purpose. This container is translucent which allows for the observation of the enzymatic reaction, and contains pure CO₂. The CO₂ hydration reaction leads to a local quick color change on the membrane from purple to yellow, where the HCAII, which is able to hydrate the CO₂, is located. After a first control assay, the membrane is subjected to a 15 min. thermal treatment at 53° C. by soaking it in a 53° C. pre-heated buffer 25 mM TAPS/100 mM Na₂SO₄ pH 8.4. This is the minimal treatment necessary to completely eliminate the signal created by the native HCAII and allows the activity of the improved mutant enzymes to be highlighted. A second developing process of the membrane as described above is performed after the thermal process, and the permanent signals from stabilized HCAII are located. For every enzymatic activity assays on membranes, a numeric camera is used in order to record all the data and facilitate their later analysis.

Example 5 Identification of Thermostabilized CA Mutants

The numeric signals were analyzed using Adobe Photoshop® software to superimpose the image of the above mentioned membrane with the corresponding Petri-dish culture and identify the clones responsible for the synthesis of a stabilized mutant HACII.

The identified clones are regrown on nutrient agar in order to validate this result. The plasmidic DNA of the clones producing stabilized HCAII is purified and the DNA sequence coding these enzymes is sequenced. (See FIGS. 17 to 26)

Example 6 Purification of Thermostabilized CA Mutants for Enzymatic Assay

The cloned recombinant CA was purified according to the following method.

The bacterial pellets were lysed with 18 mL of Lysis buffer (50 mM acetate pH 6.2 with 4 μg/mL of DNAse).

The lysed pellet solutions were centrifuged at 32 000 g during 15 minutes. The pH of the supernatant was adjusted to 6.2. The supernatants were then filtered (0.2 μm). The filtered supernatants were applied on 2 mL column of cathionic Unosphere S resin (BioRad). A first wash with 4 mL of lysis buffer followed by a second wash of 2 mL of lysis buffer with 10 mM NaCl were performed. An elution with 15 mL of lysis buffer with 75 mM NaCl was further performed.

The obtained fractions were applied onto a SDS-Page gel for the identification of fractions containing the pure CA. Then, these fractions were pooled.

The concentration of purified CA was measured at 280 nm with a molar absortion coefficient (epsilon)=5.4×10⁴ M⁻¹ cm⁻¹.

Example 7 CA Enzymatic Assay with Purified Modified CA of the Invention

0.25 mL PCR (Sarstedt) containing 250 μl of purified CA (10 μM) were incubated for 2 hours at different temperatures (55° C., 60° C., 62.5° C., 65° C. and 70° C.) in a heat bath.

The tubes were then cooled rapidly on ice for stopping the thermal denaturation. The tubes were then centrifuged (quick spin) at 13 000 RPM to obtain a pellet. The thermostability of the modified CA of the invention was determined by measuring the residual esterase activity according to the above described equation 1.

The esterase activity was measured in buffer TRIS (pH 8,0; 0.1 ionic force; 1% acetone; 0.5 mM pNPA at a temperature of 25° C.). The enzyme concentration used was 0.1 μM (40 uL of the treated solution). The esterase activity was measured by spectrophotometer at 348 nm. The results are shown in FIGS. 1 to 5.

References

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

-   1. Krebs, J., Fierke, C., 1993, J. of Biological Chemistry, Vol.     268, p. 948. -   2. Jackman, J. E., Merz, K. M., Jr., & Fierke, C. A. (1996)     Biochemistry 35, 16421 -   3. Scolnick L R, Christianson D W., 1996, Biochemistry, Vol 35, No.     51 p. 16429 -   4. Hunt. J A, Fierke C A., 1997, J. of Biological Chemistry, Vol.     272, No. 33, p. 20364

TABLE 1 Results of the residual enzymatic activity of modified carbonic anhydrase following a 2 hour treatment under high temperature conditions. ID. Mutations 55 C. 60 C. 62.5 65 C. 70 C. 0 HCAIIwt 12.6% 0.0% 0.0% 0.0% 0.0% 1 A65T 67.9% 0.0% 0.0% 0.0% 0.0% 2 F93L 62.7% 0.0% 0.0% 0.0% 0.0% 3 L100H 60.7% 0.0% 0.0% 0.0% 0.0% 4 Q136H 51.5% 0.0% 0.0% 0.0% 0.0% 5 Q136Y 62.7% 0.0% 0.0% 0.0% 0.0% 6 K153L 55.0% 0.0% 0.0% 0.0% 0.0% 7 L198M 59.7% 0.0% 0.0% 0.0% 0.0% 8 L223S 83.0% 0.0% 0.0% 0.0% 0.0% 9 L239P 70.8% 0.0% 0.0% 0.0% 0.0% 10 A247T 84.4% 0.0% 0.0% 0.0% 0.0% 1 + 3 2X 100.0% 85.0% 15.3% 0.0% 0.0% 1 + 3 + 9 3X 100.0% 95.0% 61.0% 0.0% 0.0% 1 + 3 + 6 + 9 4X 100.0% 100.0% 68.2% 6.6% 0.0% 1 + 3 + 6 + 8 + 9 5X 100.0% 100.0% 92.4% 76.4% 0.0% 1 + 3 + 6 + 8 + 9 + 10 6X 100.0% 100.0% 100.0% 88.4% 0.0% 

1. An isolated polynucleotide encoding a modified carbonic anhydrase polypeptide having increased stability under high temperature conditions compared to unmodified native carbonic anhydrase, said modified carbonic anhydrase comprising the amino acid sequence of SEQ ID NO: 1 with at least two amino acid substitutions selected from Gln136His, Leu223Ser and Leu239Pro and at least 95% identity with said SEQ ID NO:
 1. 2. The isolated polynucleotide of claim 1, wherein said modified carbonic anhydrase further comprises an amino acid substitution consisting of Ala65Thr.
 3. The isolated polynucleotide of claim 1, wherein said modified carbonic anhydrase further comprises an amino acid substitution consisting of Phe93Leu.
 4. The isolated polynucleotide of claim 1, wherein said modified carbonic anhydrase further comprises an amino acid substitution consisting of Leu100His.
 5. The isolated polynucleotide of claim 1, further comprising a substitution at position 136 consisting of Gln136Tyr, when the amino acid sequence of SEQ ID NO: 1 comprises substitutions Leu223Ser and Leu239Pro.
 6. The isolated polynucleotide of claim 1, wherein said amino acid substitutions consist of Gln136His, Leu223Ser and Leu239Pro.
 7. The isolated polynucleotide of claim 1, wherein said modified carbonic anhydrase further comprises an amino acid substitution consisting of Lys153Leu.
 8. The isolated polynucleotide of claim 1, wherein said modified carbonic anhydrase further comprises an amino acid substitution consisting of Leu197Met.
 9. The isolated polynucleotide of claim 1, wherein said modified carbonic anhydrase further comprises an amino acid substitution consisting of Ala247Thr.
 10. The isolated polynucleotide of claim 1, wherein the codon allowing the Gln136His substitution is CAT or CAC.
 11. The isolated polynucleotide of claim 1, wherein the codon allowing the Leu223Ser substitution is TCT, TCC, TCA, TCG, AGT or AGC.
 12. An expression or cloning vector comprising an isolated polynucleotide as defined in claim
 1. 13. An isolated polynucleotide encoding a modified carbonic anhydrase polypeptide having increased thermal stability compared to unmodified native carbonic anhydrase, said polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 19, and further comprising a codon allowing a Leu239Pro substitution in the encoded polypeptide.
 14. The isolated polynucleotide of claim 1, wherein the codon allowing the Leu239Pro substitution is CCT, CCC, CCA or CCG. 